LB-41

Purpose: Antiestrogen therapy is one of the most widely used and effective treatments for estrogen receptor positive breast cancer. However, de novo or acquired resistance remains a major obstacle to patient survival. Understanding the molecular mechanisms responsible for resistance is critical for improving the treatment of breast cancer. It has recently been reported that estrogen-starvation induced apoptosis of breast cancer cells is mediated by BIK, an apoptotic BH-3-only protein located primarily at the endoplasmic reticulum. As a molecular chaperone in the endoplasmic reticulum, GRP78 exhibits cytoprotective functions due to its ability to maintain ER homeostasis, suppress stress-induced apoptosis and control UPR signaling. GRP78 level is elevated in breast cancer and correlates with malignancy, metastasis, and chemotherapy resistance. The purpose of present study is to investigate the interaction between GRP78 and BIK, and the role of GRP78 in estrogen starvation-induced apoptosis in breast cancer cells.


 >Methods: Co-immunoprecipitation and GST-pulldown assays were used to probe the physical association between GRP78 and BIK. Adenoviral and plasmid expression vectors and siRNA were used to manipulate GRP78 and BIK levels. 293T cells and estrogen-dependent human breast cancer MCF-7/BUS cells were used as model systems. Cell apoptosis was examined by FACS analysis of BAX activation, mitochondrial membrane potential staining and PARP cleavage quantitation. P32 labeling was used to evaluate the effect of GRP78 on BIK phosphorylation.
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 >Results: We discovered that endogenous BIK selectively formed a complex with GRP78, probably through association with the cytosolic portion of ER transmembrane GRP78. In 293T cells, GRP78 bound to endoplasmic-reticulum targeted BIK and blocked its apoptotic activity but did not affect its phosphorylation status. In MCF-7/BUS breast cancer cells, overexpression of GRP78 inhibited estrogen-starvation induced BAX activation, mitochondrial permeability transition and consequent apoptosis. Further, knockdown of endogenous GRP78 by siRNA sensitized MCF-7/BUS cells to estrogen-starvation induced apoptosis. This effect was substantially reduced when the expression of BIK was also reduced by siRNA.
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 >Conclusion: Our results provide a new paradigm that an ER chaperone, GRP78, confers resistance to antiestrogen therapy in human breast cancer cells by inhibiting the apoptotic activity of BIK. Our studies suggest that GRP78, in addition to being a potential prognostic marker for responsiveness of human breast cancer to anti-estrogen therapy, is a novel sensitizing target for such therapy. Combination of drugs suppressing GRP78 with conventional antiestrogen therapy may represent a novel approach to improve the effectiveness of and reduce the resistance to antiestrogen therapy.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA